The overall objective of the project will be to apply the techniques of somatic cell genetics (cell hybridization, isolation and characterization of variant cell lines) to a study of the mechanism of action of insulin and insulin-like polypeptide hormones. Insulin does not stimulate DNA synthesis in mouse melanoma cells, whereas it does in mouse embryo fibroblast x melanoma hybrids. The purpose of the first part of the proposed research will be to investigate the molecular nature of the complementation that occurs in the hybrids. Initial experiments will involve a measurement of receptor sites for the insulin-like growth factor MSA on the surface of fibroblasts, the melanoma cells, and fibroblast x melanoma hybrids. In addition, a detailed examination will be undertaken of "early" and "late" events stimulated by insulin in the melanoma cells, fibroblasts and hybrids. The results of these experiments will indicate the step at which complementation for the insulin response occurs in the hybrids. Several MSA-unresponsive variants of the NIL8 hamster cell line have been isolated by a 3H-thymidine suicide technique. The second part of the project will involve a further analysis of the defect in these variants, as well as a search for new insulin and MSA-unresponsive variants of the mouse MMB cell line. The long-term aim of this study will be to delineate steps in the molecular "pathway" by which insulin and MSA stimulate growth in quiescent cells. The purpose of the third part of the project will be to map the structural gene for the human high affinity insulin receptor in human IM-9 x mouse AK-SL2 cell hybrids. The same hybrid clones will be used to examine the relationship between high affinity and low affinity insulin receptors.